Abstract
Objective:
Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced by endothelial cells. In many vessels, they are an endothelium-derived hyperpolarizing factor (EDHF). However, it is unknown whether they act as an EDHF on platelets and whether this has functional consequences.
Methods and results:
Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac4 showed a resting potential of -58+/-9 mV. Different EET regioisomers hyperpolarized platelets down to -69+/-2 mV, which was prevented by the non-specific potassium channel inhibitor charybdotoxin and by use of a blocker of calcium-activated potassium channels of large conductance (BK(Ca) channels), iberiotoxin. EETs inhibited platelet adhesion to endothelial cells under static and flow conditions. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. Stable overexpression of cytochrome P450 2C9 in EA.hy926 cells (EA.hy2C9 cells) resulted in release of EETs and a factor that hyperpolarized platelets and inhibited their adhesion to endothelial cells. These effects were again inhibited by charybdotoxin and iberiotoxin.
Conclusions:
EETs hyperpolarize platelets and inactivate them by inhibiting adhesion molecule expression and platelet adhesion to cultured endothelial cells in a membrane potential-dependent manner. They act as an EDHF on platelets and might be important mediators of the anti-adhesive properties of vascular endothelium.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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8,11,14-Eicosatrienoic Acid / analogs & derivatives*
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8,11,14-Eicosatrienoic Acid / pharmacology*
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Apamin / pharmacology
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Aryl Hydrocarbon Hydroxylases / genetics
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Aryl Hydrocarbon Hydroxylases / metabolism
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Biological Factors / physiology
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Blood Platelets / physiology*
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Cells, Cultured / cytology
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Cells, Cultured / enzymology
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Charybdotoxin / pharmacology
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Cytochrome P-450 CYP2C9
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Endothelial Cells / cytology*
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Endothelial Cells / enzymology
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Endothelium, Vascular / cytology*
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Humans
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Hydroxyeicosatetraenoic Acids / pharmacology
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Ion Channels / drug effects
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Membrane Potentials / drug effects
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Membrane Potentials / physiology*
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Peptides / pharmacology
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Platelet Adhesiveness / drug effects*
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Platelet Adhesiveness / physiology
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Platelet Aggregation / drug effects
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Platelet Aggregation / physiology
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Potassium Channels / drug effects
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Potassium Channels / physiology
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Recombinant Fusion Proteins / metabolism
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Transfection
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Umbilical Veins
Substances
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14,15-dihydroxyeicosatrienoic acid
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Biological Factors
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Hydroxyeicosatetraenoic Acids
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Ion Channels
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Peptides
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Potassium Channels
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Recombinant Fusion Proteins
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endothelium-dependent hyperpolarization factor
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Charybdotoxin
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Apamin
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11,12-epoxy-5,8,14-eicosatrienoic acid
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iberiotoxin
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5,6-epoxy-8,11,14-eicosatrienoic acid
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8,9-epoxyeicosatrienoic acid
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14,15-epoxy-5,8,11-eicosatrienoic acid
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CYP2C9 protein, human
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Cytochrome P-450 CYP2C9
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Aryl Hydrocarbon Hydroxylases
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8,11,14-Eicosatrienoic Acid